PROJECT SUMMARY/ABSTRACT Iron?sulfur clusters are found in nearly all forms of life, where they serve as essential metallocofactors for many critical biological processes. Despite decades of combined research efforts, just how the complex electronic structures of these clusters translate into their unique reactivity remains obscure. Central to this problem, especially as it pertains to clusters of high nuclearity, is characterizing the distribution of electrons at the valence level in the ground and low-lying excited states of these ubiquitous metallocofactors, a challenge requiring site- specific characterization data of individual paramagnetic Fe ions within a large exchange-coupled cluster. Herein we propose an approach toward overcoming this challenge via the site-specific labeling of synthetic and biological iron?sulfur clusters. The primary aim of this proposal is to exploit this strategy to characterize the valence electronic structure of [Fe4S4] clusters with atomic resolution. The most distinguishing feature of the electronic structures of iron?sulfur clusters is the presence of a dense manifold of many nearly-degenerate, thermally-accessible electronic states. An important consequence of this electronic structure is the availability of low-lying excited states in which the spatial arrangement of site valences differs from that of the ground state (?valence isomers?). While the ability of Fe?S clusters to sample these diverse electronic states is postulated to play a role in their reactivity, direct, experimental characterization of these electronic states is lacking. Herein, we propose a research strategy for the simultaneous measurement of the site valences of [Fe4S4] clusters in their ground and low-lying excited states via Mssbauer spectroscopy, enabled via site-selective 57Fe labeling. By performing these studies on a range of synthetic and protein-bound [Fe4S4] clusters, with variable primary and secondary coordination spheres, we can begin to delineate the structure-function relationships that underlie the dynamical valence electronic structure of these ubiquitous metallocofactors. The approaches developed herein will allow for the first characterization of the low-energy excited states of these clusters with site-specific resolution. Moreover, these approaches may be extended to other examples of biological iron?sulfur clusters, such as those in the emergent radical S-adenosylmethionine superfamily. The training plan designed under this award will equip the applicant with the skills necessary to transition to an independent career studying the roles of metals in biology, both via accomplishing the proposed research, as well as by securing the sponsorship of both junior and senior researchers in the field. Finally, the activities planned under this fellowship will benefit from the world-class research environment provided by the Massachusetts Institute of Technology.